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Endophytic fungi: a reservoir of antibacterials

Multidrug drug resistant bacteria are becoming increasingly problematic particularly in the under developed countries of the world. The most important microorganisms that have seen a geometric rise in numbers are Methicillin resistant Staphylococcus aureus, Vancomycin resistant Enterococcus faecium, Penicillin resistant Streptococcus pneumonia and multiple drug resistant tubercule bacteria to name a just few. New drug scaffolds are essential to tackle this every increasing problem. These scaffolds can be sourced from nature itself. Endophytic fungi are an important reservoir of therapeutically active compounds. This review attempts to present some data relevant to the problem. New, very specific and effective antibiotics are needed but also at an affordable price! A Herculean task for researchers all over the world! In the Asian subcontinent indigenous therapeutics that has been practiced over the centuries such as Ayurveda have been effective as “handed down data” in family generations. May need a second, third and more “in-depth investigations?”

Introduction

The last two decades have witnessed a rise in the numbers of Methicillin resistant Staphylococcus aureus (MRSA), Vancomycin resistant Enterococcus faecium (VRE) and Penicillin resistant Streptococcus pneumoniae (PRSP) and a variety of antibiotics (Menichetti, 2005). New drugs such as Linezolid and Daptomycin have already acquired resistance (Mutnick et al., 2003; Skiest, 2006). MDR- and XDR-TB (Gillespie, 2002; LoBue, 2009) are emerging global threats, being difficult to diagnose, expensive to treat and with variable results. Rice (2008) reported that the ESKAPE organism's E. faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumanii, P. aeruginosa, and Enterobacter species are the main causative agents of infections in a majority of US hospitals. To combat all these continuing developments, a search for new and novel drugs scaffolds remains the high priority activity.

Eighty five years after the discovery of Penicillin in 1929, scientists all over the world continue to investigate natural products. The novelty of structures and scaffolds, their varied bioactivities plus their abilities to act as lead molecules is immense. According to Newman and Cragg (2012), in the years 1981–2010, ~50% of all small molecules originated from natural products. Mainly antibacterial, anticancer, antiviral and antifungals compounds from natural sources such as plant, fungi and bacteria themselves. The extraordinary advantages of natural products as sources of biotherapeutics is beyond question.

Though diverse chemical compounds with equally diverse scaffolds and bioactivities have been reported from fungi over the years, the vast group still remains to be fully exploited. Out of ~1 million different fungal species only ~100,000 have been described (Hawksworth and Rossman, 1997). Dreyfuss and Chapela (1994) estimated that endophytic fungi, alone could be ~1 million. The genetic diversity of fungal endophytes may be a major factor in the discovery of novel bioactive compounds (Gunatilaka, 2006). The true potential of these endophytes is yet to be trapped.

From the first reports of isolation from the Lolium temulentum typically known as Darnel (ryegrass) by Freeman (1904), to the latest one from Antarctic moss (Melo et al., 2014), endophytic fungi have attracted the attention of botanists, chemists, ecologists, mycologists, plant pathologists and pharmacologists. It is estimated that each and every of the almost 300,000 plants that exist, hosts one or more endophyte (Strobel and Daisy, 2003). They occur everywhere, from the Arctic to Antarctic and temperate to the tropical climates. Endophytes reside in internal tissues of living plants but this association does not cause any immediate, overt, negative effects on the host plant (Bacon and White, 2000). According to Aly et al. (2011), the endophyte-plant host relationship is a balanced symbiotic continuum, ranging from mutualism through commensalism to parasitism. Many endophytic fungi remain quiescent within their hosts until it stressed or begins to undergo senescence. At this juncture the fungi may turn pathogenic (Rodriguez and Redman, 2008).

The need for novel antibacterials to combat this increasing variety of infections becomes a priority endeavor. Endophytic fungi may be an important source for such biotherapeutics like new antibacterials against Mycobacterium tuberculosis especially in poverty ridden tropical countries of Asia. Here the need could also involve a nutritional efforts to boost the immunity in the population. Many of the compounds with their host plants are shown in Table 1.

TABLE 1

Table 1. Antibacterial compounds reported from endophytic fungi.

Antibacterials from Endophytic Fungi

Compounds from Ascomycetes

Ascomycetes are an important class of fungi where there is formation of ascospores. Some genera of this class are prolific producer of bioactive metabolites. The genus Pestalotiopsis exists as an endophyte in most of the world's rainforests and is extremely biochemically diverse. Some examples of products from this group are Ambuic acid (1) and its derivative (2) (Figure 1) isolated from a Pestalotiopsis sp. of the lichen Clavaroid sp. Compounds (1) and (2) are active against S. aureus (ATCC 6538) with IC50 values of 43.9 and 27.8 μ M, respectively (the positive control Ampicillin showed an IC50 value of 1.40 μ M) (Ding et al., 2009).

Phomopsis, another important genus exists as an endophyte in most plants and is also extremely biochemically diverse. Examples of bioactive metabolites from this endophyte are Dicerandrol A (6), B (7), and C (8) (Figure 1) from Phomopsis longicolla of the mint Dicerandra frutescens. They exhibit zones of inhibition of 11, 9.5, and 8.0 mm against B. subtilis respectively and 10.8, 9.5, and 7.0 mm respectively against S. aureus when tested at 300 μ g/disc (Wagenaar and Clardy, 2001).

Phomoxanthones A (12) and B (13) (Figure 1) were obtained from Phomopsis sp. BCC 1323, of the leaf of Tectona grandis L., from the Mee Rim district of Chaingmai Province, Northern Thailand. These compounds show significant “in vitro” antitubercular activities with MICs of 0.5 and 6.25 μg/mL respectively against Mycobacterium tuberculosis H37Ra strain, in comparison to isoniazide and kanamycin sulfate (MICs of 0.050 and 2.5 μg/mL, respectively) that are used in clinics today (Isaka et al., 2001).

Phomoxanthone A (12) (Figure 1), was also isolated from a Phomopsis sp. of the stem of Costus sp. growing in the rain forest of Costa Rica. It has activity against Bacillus megaterium at a concentration of 10 mg/mL (radius of zone of inhibition of 3–4 cm) (Elsaesser et al., 2005).

3-Nitropropionic acid (35) (Figure 2) was isolated from several strains of endophytic fungus of the genus Phomopsis sp. obtained from six species of Thai medicinal plants (Table 1) from the forest areas of Chiangmai, Nakhonrachasima, and Pitsanulok Provinces of Thailand. 3-Nitropropionic acid exhibits potent activity against Mycobacterium tuberculosis H37Ra with the MIC of 3.3 μ M, but no in vitro cytotoxicity was observed toward a number of cell lines (Chomcheon et al., 2005). 3-Nitropropionic acid is known to inhibit isocitrate lyase (ICL), an enzyme required for fatty acid catabolism and virulence in M. tuberculosis (Muñoz-Elías and McKinney, 2005).

Phoma is another genus which produces diverse compounds. Here are some examples of bioactive compounds produced by this genus. Phomol (36) (Figure 3), a novel antibiotic, was isolated from a Phomopsis sp. of the medicinal plant Erythrina crista-galli. Phomol is active against Arthrobacter citreus and Corynebacterium insidiosum with MICs of 20 and 10 μ g/mL respectively (Weber et al., 2004).

3-Hydroxypropionic acid (3-HPA) (66) (Figure 5) was isolated from the mangrove endophyte Diaporthe phaseolorum, from branches of Laguncularia racemosa, growing in Bertioga, located in south eastern Brazil. 3-HPA was active against both S. aureus and Salmonella typhi at an MIC of 64 μ g/mL (Sebastianes et al., 2012).

Microsphaeropsone A (85) and Microsphaeropsone C (86) (Figure 5), were isolated from Microsphaeropsis sp. (strain 8875) from the plant Lycium intricatum, co-occurs with their putative biogenetic Anthraquinoide precursors and Citreorosein (87). From a Microsphaeropsis species (strain no. 7177) of the plant Zygophyllum fortanesii from Gomera (Spain), large amounts of Fusidienol A (88) and the known aromatic xanthones (89), were isolated. The endophyte Seimatosporium species (internal strain no. 8883) of Salsola oppositifolia from Gomera (Spain), produced 3, 4-dihydroglobosuxanthone A (90). Compounds (85–90) were active against E. coli and B. megaterium (Krohn et al., 2009).

Chlorogenic acid (142) (Figure 10) was isolated from the endophyte strain B5 a Sordariomycete sp. of Eucommia ulmoides. Eucommia ulmoides is a medicinal plant of China and one of the main sources of Chlorogenic acid. It has antibacterial, antifungal, antioxidant and antitumor activities (Chen et al., 2010).

Two new dihydroisocoumarin derivatives Aspergillumarins A (156) and B (157) (Figure 11) are produced by a marine-derived Aspergillus sp., of the mangrove Bruguiera gymnorrhiza collected from the South China Sea. Both show weak antibacterial activities against S. aureus and B. subtilis at 50 μ g/mL (Li et al., 2012).

Sanguinarine (196) (Figure 14), a benzophenanthridine alkaloid was obtained from the endophyte Fusarium proliferatum (strain BLH51) present on the leaves of Macleaya cordata of the Dabie Mountain, China. It has antibacterial, anthelmintic, and anti-inflammatory activities (Wang et al., 2014). It has antibacterial activities against the range of bacteria with MICs of 3.12–6.25 μg/mL against 15 clinical isolates of S. aureus while the MICs against of the two reference strains are 3.12 μg/mL for ATCC 25923 and 1.56 μg/mL for ATCC 33591.

The clinical isolates strains showed MIC values ranging from 31.25 to 250 μg/mL for ampicillin and 125–1000 μg/mL for ciprofloxacin. The treatment of the cells with sanguinarine induced the release of membrane-bound cell wall autolytic enzymes, which eventually resulted in lysis of the cell. Transmission electron microscopy (TEM) of MRSA treated with Sanguinarine show alterations in septa formation. The predisposition of lysis and altered morphology seen by TEM indicates that sanguinarine acts on the cytoplasmic membrane (Obiang-Obounou et al., 2011). The compound also has activity against plaque bacteria with MICs of 1–32 μg/mL for most species tested. The Electron microscopic studies of bacteria exposed to sanguinarine show that they aggregate and become morphologically irregular (Godowski, 1989).

Periconicins A (212) and B (213) (Figure 15), were isolated from an endophyte Periconia sp., from the branches of Taxus cuspidata. Periconicin A (212) has significant activity against B. subtilis, S. aureus, K. pneumoniae, and Salmonella typhimurium with MICs in the range of 3.12–12.5 μ g/mL. Periconicin B (213) has modest antibacterial activity against the same strains with MICs in the range 25–50 μ g/mL (Kim et al., 2004).

Piperine (214) (Figure 15), which was originally isolated from Piper longum, was also detected from the endophyte Periconia sp. of the same plant. Piperine has strong activity against M. tuberculosis and M. smegmetis with MICs of 1.74 and 2.62 μ g/mL respectively (Verma et al., 2011).

Fusidilactones D (233) and E (234) (Figure 17) were isolated from the endophyte, a Fusidium sp. from the leaves of Mentha arvensis growing in a meadow near Hahausen, Lower Saxony, Germany. Both compounds are weakly active against E. coli and B. megaterium (Qin et al., 2009).

Compounds Produced from Unidentified Fungi

Nonsporulating fungi form a major group of such endophytes. Khafrefungin, Arundifungin are antifungals reported from such fungi (Deshmukh and Verekar, 2012). Bostrycin (242) (Figure 18) isolated from the mangrove endophyte, no. 1403, of the South China Sea (Xu et al., 2010a), shows antibacterial activity against B. subtilis (Charudattan and Rao, 1982).

Guanacastepene A (243) (Figure 18), a novel diterpenoid produced the fungus CR115 isolated from the branch of Daphnopsis americana growing in Guanacaste, Costa Rica, may prove to belong to potentially new class of antibacterial agents with activities against MRSA and VRE (Singh et al., 2000). Guanacastepene I (244) (Figure 18), was isolated from the same fungus is active against S. aureus (Brady et al., 2001).

Anhydrofusarubin (245) (Figure 18), was isolated from the endophyte no. B77 of a mangrove tree on the South China Sea coast. Compound (245) is active against Staphylococcus aureus (ATCC27154) with a MIC of 12.5 μ g/mL (Shao et al., 2008b).

Volatile organic compounds from endophytic fungi

Strobel et al. (2001) reported at least 28 volatile organic compounds (VOC) from the xylariaceaous endophyte Muscodor albus (isolate 620), of Cinnamomum zeylanicum from Lancetilla Botanical Garden near La Ceiba, Honduras. These VOC's are mixtures of gasses of five class's viz. alcohols, acids, esters, ketones and lipids. The most effective were the esters, of which, 1-butanol, 3-methyl-acetate has the highest activity. The VOC's inhibited and killed certain bacteria, within a period of 1–3 days. Most test organisms were completely inhibited, and in fact killed. These includes Escherichia coli, Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis along with some fungal species.

Strain of Muscodor namely Muscodor crispans of Ananas ananassoides (wild pineapple) growing in the Bolivian Amazon Basin produces VOC's; namely propanoic acid, 2-methyl-; 1-butanol, 3-methyl-; 1-butanol, 3-methyl-, acetate; propanoic acid, 2-methyl-, 2-methylbutyl ester; and ethanol. The VOC's of this fungus are effective against Xanthomonas axonopodis pv. citri a citrus pathogens. The VOC's of M. crispans kill several human pathogens, including Yersinia pestis, Mycobacterium tuberculosis and Staphylococcus aureus. Muscodor crispans is only effective against the vegetative cells of Bacillus anthracis, but not against the spores. Artificial mixtures of the fungal VOC's were both inhibitory and lethal to a number of human and plant pathogens, including three drug-resistant strains of Mycobacterium tuberculosis (Mitchell et al., 2010). The mechanism of action of the VOC's of Muscodor spp. on target bacteria is unknown. A microarray study of the transcriptional response analysis of B. subtilis cells exposed to M. albus VOC's show that the expression of genes involved in DNA repair and replication increased, suggesting that VOC's induce some type of DNA damage in cells, possibly through the effect of one of the naphthalene derivatives (Mitchell et al., 2010).

Outlook

A definite, urgent and worldwide effort is needed to tackle the problems of the populations in third world and developing countries. MRSA, VRE, PRSP, ESCAPE organisms have spread through these countries over the years particularly due to immunocompromised populations. Mycobacterium tuberculosis is a major threat! and New and Novel drugs are a must!! Endophytic fungi may be an excellent source of such compounds. These organisms have a vast repertoire of diverse chemicals such as steroids, xanthones, phenols, isocoumarins, perylene derivatives, quinones, furandiones, terpenoids, depsipeptides and cytochalasins (Tan and Zou, 2001; Gunatilaka, 2006; Zhang et al., 2006; Guo et al., 2008).

A major challenge in Drug Discovery Program based on endophytic fungi lies in developing effective strategies to isolating bioactive strains. Strobel and Daisy (2003) suggested that areas of high biodiversity of endemic plant species may hold the greatest potential for endophytes with novel chemical entities. Tropical forests are some of the most bio diverse ecosystems and their leaves are “biodiversity hotspots” (Arnold and Lutzoni, 2007). The selection of plants is crucial. Those with medicinal properties should be given preference. Metabolites produced by fungi need to correlated with the plant genomics, thus allowing far better knowledge of biosynthetic pathways. This will also justify the production of metabolites rather than unproven hypotheses.

Identification of endophytic fungi using molecular analyses provides an opportunity to look for broad patterns in bioactivity not only at the genotype or strain level, but at higher taxonomic levels that may in turn assist in focusing on the association of metabolite with the plant.

The endophytic flora of the Indian subcontinent has been explored for their diversity but not enough for their bioactive metabolites. The published work is scanty (Puri et al., 2005; Deshmukh et al., 2009; Khrawar et al., 2009; Periyasamy et al., 2014). There is a need for groups from different scientific discipline (mycologist, chemist, toxicologist, and pharmacologist) to engage in this search process. Enormous natural wealth exists in the world's tropical forests, but disparity exists between developed countries with their financial resources and biodiversity rich countries with underdeveloped economy and limited funds. May be funding agencies need to look at such aspects.

The need of a more and larger collection of fungal endopytes is suggested. Bioactive metabolite metabolites from such collections could yield leads for pharmaceutical and agricultural application.

What emerges is the essential bonding of various discipline of biology and chemistry into cohesive target delivery vehicles.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The authors are extremely grateful to Dr. B. N. Ganguli, Emeritus Scientist of the CSIR, India and Chair Professor of the Agharkar Research Institute, Pune, Fellow of the Biotechnology Research Institute of India for his very careful scrutiny and suggestions of this review paper.

Rodriguez, R., and Redman, R. (2008). More than 400 million years of evolution and some plants still can't make it on their own: plant stress tolerance via fungal symbiosis. J. Exp. Bot. 59, 1109–1114. doi: 10.1093/jxb/erm342